Logarithmic confidence estimation of a ratio of binomial proportions for dependent populations.

This article investigates the logarithmic interval estimation of a ratio of two binomial proportions in dependent samples. Previous studies suggest that the confidence intervals of the difference between two correlated proportions and their ratio typically do not possess closed-form solutions. Moreover, the computation process is complex and often based on a maximum likelihood estimator, which is a biased estimator of the ratio. We look at the data from two dependent samples and explore the general problem of estimating the ratio of two proportions. Each sample is obtained in the framework of direct binomial sampling. Our goal is to demonstrate that the normal approximation for the estimation of the ratio is reliable for the construction of a confidence interval. The main characteristics of confidence estimators will be investigated by a Monte Carlo simulation. We also provide recommendations for applying the asymptotic logarithmic interval. The estimations of the coverage probability, average width, standard deviation of interval width, and index H are presented as the criteria of our judgment. The simulation studies indicate that the proposed interval performs well based on the aforementioned criteria. Finally, the confidence intervals are illustrated with three real data examples.

Inhibitory Mechanisms of Myricetin on Human and Rat Liver Cytochrome P450 Enzymes.

BACKGROUND AND OBJECTIVES: Myricetin is a flavonoid compound that is abundant in teas, red wine, berries, herbs and vegetables with a variety of pharmacological properties such as antioxidant, anti-inflammatory and anti-cancer effects. Although there are in vitro studies showing that myricetin inhibits human cytochrome P450 (CYP) 2D6 and CYP3A, the inhibitory mechanisms of myricetin on CYP enzymes are still unclear. The aim of this study was to evaluate the inhibitory effects of myricetin on human and rat CYPs, including CYP3A2/3A4, CYP2B1/2B6, CYP2C9/2C11 and CYP2D1/2D6. METHODS: This study was performed to investigate the inhibitory effects of myricetin on human CYP3A4, CYP2B6, CYP2C9, CYP2D6 and rat CYP3A2, CYP2B1, CYP2C11, CYP2D1 through the cocktail approach using ultra-performance liquid chromatography tandem mass spectrometry. Typical probe substrates were used as follows-midazolam for CYP3A2/3A4, dextromethorphan for CYP2D1/2D6, tolbutamide for CYP2C9/2C11, and bupropion for CYP2B1/2B6. RESULTS: The results of this study showed that myricetin might not be a time-dependent inhibitor. Moreover, myricetin inhibited CYP3A4 in an uncompetitive way with an inhibition constant (Ki) value of 143.1 µM. It was also a noncompetitive inhibitor of CYP2C9 and CYP2D6 with Ki values of 31.12 and 53.44 µM and a competitive inhibitor of CYP2B1 with a Ki value of 69.70 µM, as well as a mixed inhibitor of CYP3A2, CYP2C11 and CYP2D1with Ki values of 37.57, 14.88 and 17.39 µM, respectively. CONCLUSIONS: In conclusion, this study indicates that myricetin inhibited CYP3A4/3A2, CYP2C9/2C11, CYP2D6/2D1 and CYP2B1 by various mechanisms with different Ki values. Given that our experiments are established in vitro, further in vivo work is needed to confirm the interaction between myricetin and CYP enzymes, thus providing better guidance for the safe clinical use of myricetin.

Determination and pharmacokinetic study of Enasidenib in rat plasma by UPLC-MS/MS.

Enasidenib, an oral product for treating Acute Myeloid Leukemia, has been approved by FDA in Aug, 2017. In this study, we set up an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for measuring Enasidenib and imatinib (internal standard, IS), simultaneously. Enasidenib and imatinib were separated on an ACQUITY UPLC BEH C18 Column (2.1 mm × 50 mm, 1.7 µm, 132 Å). Mass detection was carried out by electrospray ionization in the position mode, and the multiple reaction monitoring transitions were m/z 474.23 → 456.17 and m/z 494.30 → 394.20 for Enasidenib and imatinib, respectively. Linearity (2 - 500 ng·mL-1, R2 > 0.999), precision and accuracy (RE < ±â€¯15%), extraction recovery (≥ 96.69%), matrix effect (≥ 96.47%) and stability (RE < ±â€¯10%) were validated which demonstrated the robustness of our method. This rapid, efficient and reliable UPLC-MS/MS method shows specificity and repeatability of Enasidenib in rat plasma and can be used in further pharmacokinetic studies.

A simple approach for monitoring business service time variation.

Control charts are effective tools for signal detection in both manufacturing processes and service processes. Much of the data in service industries comes from processes having nonnormal or unknown distributions. The commonly used Shewhart variable control charts, which depend heavily on the normality assumption, are not appropriately used here. In this paper, we propose a new asymmetric EWMA variance chart (EWMA-AV chart) and an asymmetric EWMA mean chart (EWMA-AM chart) based on two simple statistics to monitor process variance and mean shifts simultaneously. Further, we explore the sampling properties of the new monitoring statistics and calculate the average run lengths when using both the EWMA-AV chart and the EWMA-AM chart. The performance of the EWMA-AV and EWMA-AM charts and that of some existing variance and mean charts are compared. A numerical example involving nonnormal service times from the service system of a bank branch in Taiwan is used to illustrate the applications of the EWMA-AV and EWMA-AM charts and to compare them with the existing variance (or standard deviation) and mean charts. The proposed EWMA-AV chart and EWMA-AM charts show superior detection performance compared to the existing variance and mean charts. The EWMA-AV chart and EWMA-AM chart are thus recommended.

Protective effects and mechanism of total coptis alkaloids on a beta 25-35 induced learning and memory dysfunction in rats.

OBJECTIVE: To observe the effect of total coptis alkaloids (TCA) on beta -amyloid peptide (A beta 25-35) induced learning and memory dysfunction in rats, and to explore its mechanism. METHODS: Forty male Wistar rats were randomly divided into four groups: the control group, the model group, the TCA low dose (60 mg/kg) group and the TCA high dose (120 mg/kg) group, 10 in each. A beta 25-35 (5microl, 2 microg/microl) was injected into bilateral hippocampi of each rat to induce learning and memory dysfunction. TCA were administered through intragavage for consecutive 15 days. Morris Water Maze test was used to assess the impairment of learning and memory; concentration of malondialdehyde (MDA) in cerebral cortex was determined by thiobarbituric acid reactive substance to indicate the level of lipid peroxidation in brain tissues; activity of manganese-superoxide dismutase (Mn-SOD) in cerebral cortex was determined by xanthine-oxidase to indicate the activity of the enzyme; and NF- kappa B protein expression in cerebral cortex was measured by SP immunohistochemistry. RESULTS: (1) Morris Water Maze test showed that, during the 4 consecutive days of acquisition trials, the rats in the model group took longer latency and searching distance than those in the control group (P<0.01), which could be shortened by high dose TCA (P<0.05); during the spatial probe trial on the fifth day, the rats in the model group took shorter searching time and distance on the previous flat area than those in the control group (P<0.01), which could be prolonged after TCA treatment (for low dose group, P<0.05; for high dose group, P<0.01). (2) Analysis of cerebral cortical tissues showed that, compared with the control group, MDA level got significantly increased and Mn-SOD activity decreased in the model group (both P<0.01). After having been treated with TCA, the MDA level got significantly decreased (P<0.05 and P<0.01 respectively for low and high dose group), while relative increase of Mn-SOD activity only appeared in high dose group (P<0.05). (3) Immunohistochemistry analysis showed the protein expression of NF- kappa B got significantly increased after modeling, while high dose TCA can significantly inhibit it. CONCLUSION: TCA could improve A beta 25-35 induced dysfunction of learning and memory in rats, and its protective mechanism is associated with its actions in decreasing MDA level, increasing Mn-SOD activity and inhibiting the expression of NF-kappa B in cerebral cortex.

[To study the effects of local co-transfection vascular endothelial growth factor165 and tissue-type plasminogen activator genes on inhibiting intimal hyperplasia after operation injury artery in rabbits].

OBJECTIVE: To observe the effects of local co-transfection vascular endothelial growth factor165 (VEGF165) and tissue-type plasminogen activator genes on inhibiting intimal hyperplasia and restenosis in rabbits artery after operation injury and possible mechanisms. METHODS: Micrology operation injury was used to establish the model of intimal injury of right external iliac artery in rabbits. To select 120 male New Zealand rabbits and were randomly divided into 3 groups (n = 40, in each group): Group A (physiological brine control group), Group B (pBudCE4.1 group), Group C (pBudCE4.1/VEGF165-tPA group). The vas-wall of micrology operation injury were infused respectively physiological brine, pBudCE4.1 and pBudCE4.1/VEGF165-tPA transfection solution by micro-injector. Each group were divided into 5 subgroups (n = 8, in each subgroup) randomly according to the sacrifice times (2 d, 1 week, 2 week, 4 week and 8 week after operation). The injured vascular specimen were harvested for pathology test, electric microscopy study, reverse transcription-PCR examining and immunochemistry detecting. RESULTS: The intimal area and narrow ratio of vases in Group C at every time point after operation were significantly lessened than that in Group A and Group B (P < 0.01). The narrow ratio of vases in Group C at 8 week after operation were decreased respectively by 57.9% and 59.0% than that in Group A and B. The expression of VEGF165 mRNA in Group C were increased significantly than that in Group A and B at every time point after operation (P < 0.01), the expression reached the peak at 1 week and continued to 4 week after operation. Immunohistochemical identified that tPA positive cell in Group C were significantly increased than that in Group A and B (P < 0.01) at every time point after operation. CONCLUSION: Local co-transfection VEGF165 and tPA genes could restrain intimal hyperplasia and restenosis of vas, which lay a foundation for future multi-gene therapy of vascular intimal hyperplasia.

Protective effect of ecdysterone on PC12 cells cytotoxicity induced by beta-amyloid25-35.

OBJECTIVE: To examine the protective effect of ecdysterone (ECR) against beta-amyloid peptide fragment(25-35) (Abeta(25-35))-induced PC12 cells cytotoxicity, and to further explore its mechanism. METHODS: Experimental PC12 cells were divided into the Abeta group (treated by Abeta(25-35) 100 micromol/L), the blank group (untreated), the positive control group (treated by Vit E 100 micromol/L after induction) and the ECR treated groups (treated by ECR with different concentrations of 1, 50 and 100 micromol/L). The damaged and survival condition of PC12 cells in various groups was monitored by lactate dehydrogenase (LDH) release and MTT assay. The content of malondialdehyde (MDA) was measured by fluorometric assay to indicate the lipid peroxidation. And the antioxidant enzymes activities in PC12 cells, including superoxide dismutases (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), were detected respectively. RESULTS: After PC12 cells were treated with Abeta(25-35) (100 micromol/L) for 24 hrs, they revealed a great decrease in MTT absorbance and activity of antioxidant enzymes, including SOD, CAT and GSH-Px as well as a significant increase of LDH activity and MDA content in PC12 cells (P < 0.01). When the cells was pretreated with 1-100 micromol/L ECR for 24 hrs before Abeta(25-35) treatment, the above-mentioned cytotoxic effect of Abeta(25-35) could be significantly attenuated dose-dependently, for ECR 50 micromol/L, P < 0.05 and for ECR 100 micromol/L, P < 0.01. Moreover, ECR also showed significant inhibition on the Abeta(25-35) induced decrease of SOD and GSH-Px activity, but not on that of CAT. CONCLUSION: ECR could protect PC12 cells from cytotoxicity of Abeta(25-35), and the protective mechanism might be related to the increase of SOD and GSH-Px activities and the decrease of MDA resulting from the ECR-pretreatment.

Construction and biological activities of human tPA eukaryotic expression plasmid.

BACKGROUND: Tissue-type plasminogen activator(tPA), which is highly efficient and specific in resolution of thrombus, could significantly improve the survival rate and life-quality of the patients with thrombosis. This study was designed to clone human tPA gene, construct eukaryotic expression plasmid pcDNA3.1(+)/tPA, and evaluate their biological activities. METHODS: The tPA gene was obtained from dead human heart tissue with reverse transcriptase-polymerase chain reaction (RT-PCR). It was cloned to eukaryotic expression plasmid pcDNA3.1(+) by recombination strategy. The eukaryotic expression plasmid pcDNA3.1(+)/tPA was identified by restriction enzyme digestion and sequenced. The pcDNA3.1(+)/tPA was transfected into vascular smooth muscle cell (VSMC) by using lipofection. The tPA expression level was detected by Northern blot and dot blot, and the protein biological activities of tPA were detected by the fibrin plate technique. RESULTS: The tPA gene was cloned and pcDNA3.1(+)/tPA was constructed. The tPA expression levels of mRNA and protein were highly increased after pcDNA3.1(+)/tPA transfected into VSMC, and the expression of tPA protein showed evident biological activities. CONCLUSIONS: The present study has laid a foundation for further animal experiment in treating thrombus in transplanted organ by tPA gene transfection.

Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles.

BACKGROUND: The highly specific vascular endothelial growth factor (VEGF) induces the growth of vascular endothelial cell. This study was to construct the eukaryotic expression plasmid of vascular endothelial growth factor165 (VEGF165) and observe its expression in vascular smooth muscles (VSMCs). METHODS: The primers were designed and synthesized according to the gene sequences of human VEGF165. The VEGF165 gene was obtained from umbilic artery tissue by the method of RT-PCR, then it was cloned to eukaryotic expression plasmid pBudCE4.1 by recombination strategy. The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion, and was sequenced. The pBudCE4.1/VEGF165 was transfected into VSMCs by using lipofection. The VEGF165 expression of mRNA and protein was detected by RT-PCR and Western blot respectively. RESULTS: VEGF165 was shown about 576bp by RT-PCR. Sequencing revealed the amplified VEGF165 gene was identical with that in the GeneBank. Restrictive enzyme (Hind III, Bam HI) digestion analysis showed that recombinant expression plasmid pBudCE4.1/tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by RT-PCR and Western blot. CONCLUSIONS: The recombinant eukaryotic expression plasmid pBudCE4.1/VEGF165 has been successfully constructed and expressed in transformed VSMCs. The present study has laid a foundation for VEGF165 gene therapy of vascular stenosis in the transplant organ.

[Effect of ecdysterone on the expression of c-fos in the brain of rats induced by microinjection beta-AP25-35 into the hippocampus].

AIM: To observe the behavior in learning and memory and the expression of c-fos gene from the brain of rats induced by beta-AP25-35, and the intervention of ecdysterone, in order to explore the protective mechanism of ecdysterone on the dysfunction of learning and memory of the rat induced by beta-AP25-35. METHODS: Microinjection of beta-AP25-35 into hippocampus induced learning and memory dysfunction of rats. The learning and memory of rats were observed by Morris Water Maze. The expression of c-fos gene in the brain was detected by immunohistochemistry. RESULTS: The results of Morris Water Maze showed that after rats were microinjected beta-AP25-35 into hippocampus, the rats in model group took longer latency and searching distance compared with the ones in control group (P < 0.01), and the rats in treated group (ECR 4 mg x kg(-1), ECR 8 mg x kg(-1) and nimodipine 7.2 mg x kg(-1)) took shorter latency and searching distance, especially the ECR 8 mg kg(-1) group (P < 0.01). At the same time, after the 5 days training, there was a higher expression of c-fos in hippocampus and cortex from the rats in control group than that in model group (P < 0.01), but in the treated group, there was a relatively higher expression of c-fos, especially the ECR 8 mg x kg(-1) group (P < 0.01). CONCLUSION: Microinjection of beta-AP25-35 into the rat hippocampus resulted in dysfunction of learning and memory. Ecdysterone was shown to improve the learning and memory of the rats and increase the expression of c-fos. Increasing the expression of c-fos is probably one of the most molecular mechanism of its protection.

[The effects of genistein on epidermal growth factor receptor mediated signal transduction pathway in human ovarian carcinoma cells lines SKOV3 and its xenograft in nude mice].

OBJECTIVE: It was reported previously that genistein could inhibit proliferation of human ovarian carcinoma cell line SKOV(3), but mechanism was not clear. There is a close relationship between EGFR mediated signal transduction pathway and the development of ovarian tumor. This study aimed to investigate the effects of genistein on the EGFR mediated signal transduction pathway and to clarify its mechanisms of proliferation inhibition on human ovarian carcinoma cell line SKOV(3) and its xenograft in nude mice. METHODS: The expression of c-erbB-2 protein was determined using immunocytochemistry. The expressions of c-jun and c-fos protein were determined using Western blotting. The expression of c-erbB-2, c-raf-1, c-jun and c-fos mRNA were tested by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression of c-erbB-2, c-raf-1 and its downstream gene c-jun and c-fos were decreased at mRNA level in the 20 micromol/L genistein group. The expression of c-erbB-2 protein were decreased, its average absorbency (A) were decreased after treatment of SKOV(3) with 20 micromol/L genistein for 48 h, reached at 0.42 +/- 0.02 (P < 0.05). Western blotting demonstrated that the expressions of c-jun and c-fos protein were decreased gradually after being treated with 20 micromol/L genistein for 12 - 72 h. CONCLUSIONS: Genistein could down-regulate the expression of two key genes, c-erbB-2 and c-raf-1 at mRNA and protein level in the EGFR mediated signal transduction pathway, and down-regulate the expression of its downstream nuclear transcription factors c-jun and c-fos at mRNA and protein level. It is suggested that interfering the expressions of some key signal molecules in EGFR mediated signal transduction system by genistein may account for its molecular foundation of proliferation inhibition in ovarian carcinoma.

[Inhibition effects of c-erbB-2 and c-raf-1 antisense oligodeoxynucleotides combined transfection on the human ovarian carcinoma transplanted subcutaneously in nude mice].

OBJECTIVE: To investigate the inhibition effects of c-erbB-2 and c-raf-1 antisense oligodeoxynucleotides (ASODN) combined transfection on the human ovarian epithelial cancer transplanted subcutaneously in nude mice. METHODS: There were 7 groups: normal control group, c-erbB-2 sense observed group, c-raf-1 sense observed group, c-erbB-2 antisense observed group, c-raf-1 antisense observed group, whole dose combined group, half dose combined group. Human ovarian epithelial cancer cells SKOV3 were treated by different oligodeoxynucleotides, then transplanted subcutaneously in nude mice, respectively. The changes of tumor volume were observed and the tumor growth inhibitory rate was calculated. RESULTS: There was no difference between sense observed group and normal control group. There was a larger growth inhibitory rate in whole -dose combined group and half -dose combined group, the first time that can be detected was 13.7 days and 15.2 days, and the maximum tumor growth inhibitory rates were 61.1% and 71.3%, respectively. CONCLUSIONS: The results suggested that ASODN combined transfection can inhibit the tumorigenesis of ovarian epithelial cancer cells in nude mice, it may be a more useful gene therapy for the ovarian epithelial carcinoma.